There are many instances when it is required to separate one compound, such as a contaminant or a desired molecule, from a liquid or from other solid materials. Charge-charge based interactions are used in a number of fields to capture and hence separate charged or chargeable compounds.
In the detergent industry, methods and detergents are available to separate compounds such as soil and dirt from textile fabrics. A frequently encountered example is the laundry cleaning industry, where charged conditioners and detergents are commonly incorporated in washing powders for separating soil from the washed materials. Such charged conditioners, also known as benefit agents, are used either to directly release soil from the textile fibres; or to modify the fibres to facilitate the cleaning process; by the use of attractive forces between the soil and conditioner. To improve their effectiveness, it has been suggested to provide conditioners in a form wherein they are substituted onto another chemical moiety, which increases their affinity for the compound to be removed.
WO 03/040279 relates to polymers for laundry applications, and more specifically to the use of substituted polysaccharide structures for promoting soil release during the laundry of a textile fabric. The suitable polysaccharides include polysaccharides with a degree of polymerisation over 40, preferably in the range of 50-100 000. The polysaccharide structures disclosed have been substituted with an alkyl group, such as hydroxyalkyl, carboxyalkyl or sulphoalkyl or a salt thereof, coupled to the polysaccharide via ester or ether linkage. The average degree of substitution i.e. the average substitution of the functional groups on the repeating sugar unit is preferably from 0.1-3, more preferably from 0.1-1. According to WO 03/040279, polysaccharides can be used which have an α- or β-linked backbone, preferably a β-1,4-linked backbone. Since cellulose-based materials have been recognised in the art to adhere to cotton fibres, the preferred polysaccharide is cellulose.
In the chemical and biotech field, target compounds such as drug or drug candidates usually need to be separated from contaminating species originating from the process of manufacture. For example, a protein drug or drug candidate produced by expression of recombinant host cells will need to be separated e.g. from the host cells and possibly cell debris, other host cell proteins, DNA, RNA, and residues from the fermentation broth such as salts. Due to its versatility and sensitivity to the target compounds, chromatography is involved as at least one step in many of the currently used biotech purification schemes. The term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components of the sample.
The stationary phase in chromatography is comprised of a solid carrier to which ligands, which are functional groups capable of interaction with the target compound, have been coupled. Consequently, the ligands will impart to the carrier the ability to effect the separation, identification, and/or purification of molecules of interest. Liquid chromatography methods are commonly named after the interaction principle utilised to separate compounds. For example, ion exchange chromatography is based on charge-charge interactions; hydrophobic interaction chromatography (HIC) utilises hydrophobic interactions; and affinity chromatography is based on specific biological affinities.
As is well known, ion exchange is based on the reversible interaction between a charged target compound and an oppositely charged chromatography matrix. The elution is most commonly performed by increasing the salt concentration, but changing the pH is equally possible. Ion-exchangers are divided into cation-exchangers, wherein a negatively charged chromatography matrix is used to adsorb a positively charged target compound; and anion-exchangers, wherein a positively charged chromatography matrix is used to adsorb a negatively charged target compound. The term “strong” ion exchanger is used for an ion-exchanger which is charged over broad pH intervals, while a “weak” ion-exchanger is chargeable at certain pH values. One commonly used strong cation exchanger comprises sulphonate ligands, known as S groups. In some cases, such cation exchangers are named by the group formed by the functional group and its linker to the carrier; for example SP cation exchangers wherein the S groups are linked by propyl to the carrier.
The properties of the carrier to which the ligands have been coupled will also affect the separation properties of a chromatography matrix. Depending on the intended mode of chromatography, carriers that are substantially hydrophilic or hydrophobic may be preferred. A further consideration of the carrier is the ease of which it is functionalized. Depending on the chemistry used for coupling ligands, the carrier may be activated i.e. transformed into a more reactive form. Such activation methods are well known in this field, such as allylation of the hydroxyl groups of a hydrophilic carrier, such as dextran or agarose. Covalent ligand attachment is typically achieved by the use of reactive functionalities on the solid support matrix such as hydroxyl, carboxyl, thiol, amino groups, and the like. In order to enhance the binding capacity of the matrix, a linking arm known simply as a linker is often provided between the ligand and carrier. Such linkers will physically distance the ligand from the carrier, whereby the target compound is allowed to interact with the ligand with minimal interference from the matrix. However, the use of linkers in the synthesis of chromatography matrices requires the use of a functional reagent having at least one functional group capable of reacting with a functional group on the surface of the matrix to form a covalent bond therewith; and at least one functional group capable of reacting with a functional group on the ligand to form a covalent bond therewith.
U.S. Pat. No. 5,789,578 (Massey University) relates to methods for the preparation of chromatography matrices comprising a support matrix having ligands capable of binding a target compound covalently attached thereto through a linking group comprising sulfide, sulfoxide, or sulfone functionality. Bisulphite is used as a reagent to provide S groups. More specifically, U.S. Pat. No. 5,789,578 uses allyl glycidyl ether, allyl halide or propargyl halide and conventional methods in the presence of a base to provide a carrier having ethylenically unsaturated entities pendent thereto. Specifically, the halide or glycidyl group reacts with matrix hydroxyl groups at alkaline pH. Under these conditions, the allyl group is expected to have limited reactivity with the matrix or water used in the reaction solution. The ethylenically unsaturated group is then reacted under free radical conditions with a thiol-containing ligand to provide for covalent linkage thereof.
However, the introduction of allyl groups and subsequent coupling of S groups as disclosed in the above-discussed U.S. Pat. No. 5,789,578 will leave a fraction of the allyl groups unreacted on the carrier while another fraction will be subject to the introduction of vicinal sulphonate-sulphinate groups. In addition, the activated reagents will also involve the risk of undesired cross-linking reactions. Obviously, any step added to a process of for example drug manufacture will make the process more costly, and it is therefore a general aim in chemical processing to make any process as brief as possible.
Thus, there is a clear need in this field of improved methods which allows functionalization of polysaccharide carriers in a faster and more robust way, preferably avoiding one or more of the above-discussed disadvantages.